Ultrasound contrast agents containing microbubbles of perfluoracarbon gasses

ABSTRACT

Disclosed herein are agents for enhancing the contrast in an ultrasound image. These agents are extremely small bubbles, or “microbubbles,” comprised of specially selected gases. The microbubbles described herein exhibit long life spans in solution and may be produced at a size small enough to traverse the lungs, thus enabling improved ultrasound imaging of the cardiovascular system and other vital organs. Also disclosed herein is a method for selecting gases from which contrast agents may be produced. The method is based on calculations using inherent physical properties of gases and describes a means to associate the properties of a gas with the time for dissolution of microbubbles comprised of the gas.

RELATED APPLICATIONS

[0001] This application is a continuation in part of U.S. applicationSer. No. 07/893,657 filed Jun. 5, 1992, which is a continuation in partof U.S. application Ser. No. 07/761,311 filed Sep. 17, 1991.

DESCRIPTION

[0002] This invention relates to agents that enhance the contrast in anultrasound image generated for use in medical diagnosis. Thecontrast-enhancing media disclosed herein are comprised of extremelysmall gas bubbles which are present in a solution that is infused intothe body during or just before an ultrasound image is generated. Thisinvention is also directed to a method for enhancing such images byselecting gases from which a collection of free gas microbubbles can beprepared that have novel and superior properties. These microbubbles,composed of the gases whose selection is enabled by the process of thisinvention, may be extremely small in size and yet survive in thebloodstream long enough to allow contrast-enhanced imaging of parts ofthe cardiovascular system, peripheral vascular system, and vital organspreviously believed to be inaccessible to free gas microbubbles.

BACKGROUND

[0003] When using ultrasound to obtain an image of the internal organsand structures of a human or animal, ultrasound waves—waves of soundenergy at a frequency above that discernable by the human ear—arereflected as they pass through the body. Different types of body tissuereflect the ultrasound waves differently and the reflections, oftenaptly described as “echoes,” that are produced by the ultrasound wavesreflecting off different internal structures are detected and convertedelectronically into a visual display. This display may prove invaluableto a physician or other diagnostician in several ways, includingevaluating the progression of cardiovascular disease or the existence ornature of a tumor.

[0004] For some medical conditions, obtaining a useful image of theorgan or structure of interest is especially difficult because thedetails of the structure may not be adequately discernible from thesurrounding tissue in an ultrasound image produced by the reflection ofultrasound waves absent a contrast-enhancing agent. Additionally,traditional ultrasound images are notoriously poor in quality andresolution. For these reasons, detection and observation of certainphysiological conditions may be substantially improved by enhancing thecontrast in an ultrasound image by infusing an agent into an organ orother structure of interest. In other cases, detection of the movementof the contrast-enhancing agent itself is particularly important. Forexample, a distinct blood flow pattern that is known to result fromparticular cardiovascular abnormalities may only be discernible byinfusing a contrasting agent into the bloodstream and observing thedynamics of the blood flow.

[0005] Medical researchers have made extensive investigation into theuse of solids, gases and liquids in an attempt to discover ultrasoundcontrast-enhancing agents suitable for particular diagnostic purposes.Composite substances such as gelatin encapsulated microbubbles,gas-incorporated liposomes, sonicated partially denatured proteins andemulsions containing highly fluorinated organic compounds have also beenstudied in an attempt to develop an agent that has certain idealqualities, primarily, stability in the body and the ability to providesignificantly enhanced contrast in an ultrasound image.

[0006] Small bubbles of a gas, termed “microbubbles,” are readilydetected in an image produced using standard ultrasound imagingtechniques. When infused into the bloodstream or a particular site inthe body, microbubbles enhance the contrast between the regioncontaining the microbubbles and the surrounding tissue.

[0007] A substantial amount of the research effort directed atcontrast-enhancing agents has focused on the use of extremely small gasbubbles. Investigators have long known that free gas bubbles provide ahighly effective contrast agent because a gas bubble has unique physicalcharacteristics that affect ultrasound energy as it is directed throughthe body. The advantages offered by free gas bubbles as opposed toliquid or solid agents that exhibit contrast enhancement is described indetail below in the context of the discussion of ultrasound diagnostictechniques.

[0008] Despite the known advantages, however, the rapid dissolution offree gas bubbles in solutions such as blood or many aqueous intravenoussolutions, severely limits their use as an ultrasound contrast-enhancingagent. The most important limitations are the size of the microbubbleand the length of time that a microbubble will exist before dissolvinginto the solution.

[0009] Examining the size requirements for microbubbles more closely,the gas bubbles must, of course, be sufficiently small that a suspensionof the bubbles does not carry the risk of embolism to the organism inwhich they are infused. At the same time, extremely small free gasbubbles composed of the gases generally used in ultrasound contrastimaging dissolve into solution so rapidly that their image-enhancingcapability exists only immediately proximate to the infusion site. Anadditional obstacle exists for ultrasound imaging of the cardiovascularsystem. Medical researchers have studied the time required formicrobubbles composed of ordinary air, pure nitrogen, pure oxygen, orcarbon dioxide, to dissolve into solution. Microbubbles of these gasesthat are sufficiently small to be able to pass through the lungs andreach the left heart, less than about 8 microns in diameter, have a lifespan of less than approximately 0.25 seconds. Meltzer, R. S., Tickner,E. G., Popp, R. L., “Why Do the Lungs Clear Ultrasonic Contrast?”Ultrasound in Medicine and Biology, Vol. 6, p.263, 267 (1980). Since ittakes over 2 seconds for blood to pass through the lungs, microbubblesof these gases would be fully dissolved during passage through the lungsand would never reach the left heart. Ibid. Primarily because of thistradeoff between bubble size and life span, many researchers concludedthat free gas microbubbles were not useful as a contrast-enhancing agentfor ultrasound diagnosis of certain parts of the cardiovascular system.

[0010] However, the ultrasound contrast-enhancing media described hereincomprises microbubbles, composed of the biocompatible gases whoseselection is also provided by this invention, that are sufficientlysmall that they pass through the pulmonary capillary diameter ofapproximately 8 microns and thereby allow contrast-enhanced ultrasounddiagnosis of the left chambers of the heart. The free gas microbubblessurvive in the bloodstream long enough that they may be peripherallyintravenously infused, travel through the right heart, through thelungs, and into the left cardiac chambers without dissolving intosolution. Additionally, certain of these media have extremely longpersistence in solution and will enable contrast-enhancement of manyother organs and structures.

[0011] This invention overcomes many of the inherent limitations thoughtto exist with the use of free gas microbubbles by providing, in part, amethod for selecting special gases based on particular physical criteriasuch that microbubbles composed of these gases do not suffer from thesame limitations as the microbubbles previously investigated. Therefore,it has been discovered that the ultrasound contrast-enhancing mediadescribed herein comprising a composition of microbubbles produced usinga biocompatible gas or combination of gases selected by the physical andchemical parameters disclosed herein can exist for a sufficient lengthof time and be of sufficiently small size that their stability in thebloodstream allows enhanced ultrasound contrast imaging of particularstructures in the body previously thought inaccessible to free gasmicrobubbles.

[0012] By using the term “biocompatible gas” I mean a chemical entitywhich is capable of performing its functions within or upon a livingorganism in an acceptable manner, without undue toxicity orphysiological or pharmacological effects, and which is, at thetemperature of the living organism, in a state of matter distinguishedfrom the solid or liquid states by very low density and viscosity,relatively great expansion and contraction with changes in pressure andtemperature, and the spontaneous tendency to become distributeduniformly throughout any container. The following Table contains theassumed body temperatures for various living organisms: RectalTemperature Organism (degree Fahrenheit) Swine (Sus Scrofa) 101.5-102.5Sheep (Ovis sp.) 101-103 Rabbit (Oryctolaqus cuniculus)   102-103.5 Rat(Tattus morvegicus)  99.5-100.6 Monkey (Macaca mulatta) 101-102 Mouse(Mus Musculus)  98-101 Goat (Capra hircus) 101-103 Guinea pig (Caviaporcellus) 102-104 Hamster (Mesocricetus sp.) 101-103 Ham (Homo sapiens) 98.6-100.4 Horse (Equus sp.)   101-102.5 Dog (Canin familiaris) 101-102Baboon (Papio)  98-100 Cat (Felis catus) 101-102 Cattle (Bos taurus)101.5-102.5 Chimpanzee (Pan)  96-100

Techniques For Measuring Ultrasound Contrast-Enhancement Phenomena

[0013] To more fully appreciate the subject matter of the presentinvention, it is useful to describe what is presently known about thetechnology of ultrasound imaging and to review the search for improvedultrasound contrast-enhancing agents in that light.

[0014] Materials that are useful as ultrasound contrast agents operateby having an effect on ultrasound waves as they pass through the bodyand are reflected to create the image from which a medical diagnosis ismade. In an attempt to develop an efficient image-contrast agent, oneskilled in the art recognizes that different types of substances affectultrasound waves in different ways and to varying degrees. Moreover,certain of the effects caused by contrast-enhancing agents are morereadily measured and observed than others. Thus, in selecting an idealcomposition for a contrast-enhancing agent, one would prefer thesubstance that has the most dramatic effect on the ultrasound wave as itpasses through the body. Also, the cffect on the ultrasound wave shouldbe easily measured. There are three main contrast-enhancing effectswhich can be seen in an ultrasound image: backscatter, beam attenuation,and speed of sound differential. Each of these effects will be describedin turn.

[0015] A. Backscatter

[0016] When an ultrasound wave that is passing through the bodyencounters a structure, such as an organ or other body tissue, thestructure reflects a portion of the ultrasound wave. Differentstructures within the body reflect ultrasound energy in different waysand in varying strengths. This reflected energy is detected and used togenerate an image of the structures through which the ultrasound wavehas passed. The term “backscatter” refers to the phenomena in whichultrasound energy is scattered back towards the source by a substancewith certain physical properties.

[0017] It has long been recognized that the contrast observed in anultrasound image may be enhanced by the presence of substances known tocause a large amount of backscatter. When such a substance isadministered to a distinct part of the body, the contrast between theultrasound image of this part of the body and the surrounding tissuesnot containing the substance is enhanced. It is well understood that,due to their physical properties, different substances cause backscatterin varying degrees. Accordingly, the search for contrast-enhancingagents has. focused on substances that are stable and non-toxic and thatexhibit the maximum amount of backscatter.

[0018] Making certain assumptions about the way a substance reflectsultrasound energy, mathematical formulae have been developed thatdescribe the backscatter phenomenon. Working with these formulae, askilled researcher can estimate the ability of gas, liquid, and solidcontrast-enhancing agents to cause backscatter and the degree to which aparticular substance causes measurable backscatter can be compared withother substances based on the physical characteristics known to causethe backscatter phenomenon. As a simple example, the ability ofsubstance A to cause backscatter will be greater than substance B, if,all other factors being equal, substance A is larger than substance B.Thus, when both substances are encountered by an ultrasound wave, thelarger substance scatters a greater amount of the ultrasound wave.

[0019] The capability of a substance to cause backscatter of ultrasoundenergy also depends on other characteristics of the substance such asits ability to be compressed. Of particular importance is the dramaticincrease in backscatter caused by gas bubbles due to the bubbleresonance phenomenon which is described below. When examining differentsubstances, it is useful to compare one particular measure of theability of a substance to cause backscatter known as the “scatteringcross-section.”

[0020] The scattering cross-section of a particular substance isproportional to the radius of the scatterer, and also depends on thewavelength of the ultrasound energy and on other physical properties ofthe substance, J. Ophir and K. J. Parker, Contrast Agents in DiagnosticUltrasound. Ultrasound in Medicine & Biology, vol. IS, n. 4, p. 319, 323(1989).

[0021] The scattering cross-section of a small scatterer, a, can bedetermined by a known equation:$\sigma = {\left\lbrack {\frac{4}{9}\pi \quad {a^{2}({ka})}^{4}} \right\rbrack \quad\left\lbrack {{\frac{\kappa_{s} - \kappa}{\kappa}}^{2} + {\frac{1}{3}{\frac{3\left( {\rho_{s} - \rho} \right)}{{2\rho_{s}} - \rho}}^{2}}} \right\rbrack}$

[0022] where κ=2π/λ, where λ is the wavelength; a=the radius of thescatterer; κ_(s)=adiabatic compressibility of the scatterer; κ=adiabaticcompressibility of the medium in which the scatterer exists,ρ_(s)=density of the scatterers and ρ=the density of the medium in whichthe scatterer exists. P. M. Morse and K. U. Ingard, TheoreticalAcoustics. p. 427, McGraw Hill, N.Y. (1968).

[0023] In evaluating the utility of different substances as imagecontrasting agents, one can use this equation to determine which agentswill have the higher scattering cross-section and, accordingly, whichagents will provide the greatest contrast in an ultrasound image.

[0024] Referring to the above equation, the first bracketed quantity inthe above equation can be assumed to be constant for the purpose ofcomparing solid, liquid and gaseous scatterers. It can be assumed thatthe compressibility of a solid particle is much less than that of thesurrounding medium and that the density of the particle is much greater.Using this assumption, the scattering cross section of a solid particlecontrast-enhancing agent has been estimated as 1.75. Ophir and Parker,supra, at 325.

[0025] For a pure liquid scatterer, the adiabatic compressibility anddensity of the scatterer κ_(s) and the surrounding medium κ are likelyto be approximately equal which would, from the above equation, yieldthe result that liquids would have a scattering cross-section of zero.However, liquids may exhibit some backscatter if large volumes of aliquid agent are present presumably because the term a in the firstbracketed quantity in the above equation may become sufficiently large.For example, if a liquid agent passes from a very small vessel to a verylarge one such that the liquid occupies substantially all of the vesselthe liquid may exhibit measurable backscatter. Nevertheless, in light ofthe above equation and the following, it is appreciated by those skilledin the art that pure liquids are relatively inefficient scattererscompared to free gas microbubbles.

[0026] It is known that changes in the acoustic properties of asubstance are pronounced at the interface between two phases, i.e.,liquid/gas, because the reflection characteristics of an ultrasound wavechange markedly at this interface. Additionally, the scattercross-section of a gas is substantially different than a liquid orsolid, in part, because a gas bubble can be compressed to a much greaterdegree than a liquid or solid. The physical characteristics of gasbubbles in solution are known and standard values for compressibilityand density figures for ordinary air can be used in the above equation.Using these standard values, the result for the second bracketed termalone in the above equation is approximately 10¹⁴, Ophir and Parkersupra, at 325, with the total scattering cross section varying as theradius a of the bubble varies. Moreover, free gas bubbles in a liquidexhibit oscillatory motion such that, at certain frequencies, gasbubbles will resonate at a frequency near that of the ultrasound wavescommonly used in medical imaging. As a result, the scatteringcross-section of a gas bubble can be over a thousand times larger thanits physical size.

[0027] Therefore, it is recognized that-gas micro-bubbles are superiorscatterers of ultrasound energy and would be an ideal contrast-enhancingagent if the obstacle of their rapid dissolution into solution could beovercome.

[0028] B. Beam Attenuation

[0029] Another effect which can be observed from the presence of certainsolid contrast-enhancing agents, is the attenuation of the ultrasoundwave. Image contrast has been observed in conventional imaging due tolocalized attenuation differences between certain tissue types. K. J.Parker and R. C. Wang, “Measurement of Ultrasonic Attenuation WithinRegions selected from B-Scan Images,” IEEE Trans. Biomed. Enar. BME30(8), p. 431-37 (1983); K. J. Parker, R. C. Wang, and R. M. Lerner,“Attenuation of Ultrasound Magnitude and Frequency Dependence for TissueCharacterization,” Radiology, 153(3), p. 785-88 (1984). It has beenhypothesized that measurements of the attenuation of a region of tissuetaken before and after infusion of an agent may yield an enhanced image.However, techniques based on attenuation contrast as a means to measurethe contrast enhancement of a liquid agent are not well-developed and,even if fully developed, may suffer from limitations as to the internalorgans or structures with which this technique can be used. For example,it is unlikely that a loss of attenuation due to liquid contrast agentscould be observed in the image of the cardiovascular system because ofthe high volume of liquid contrast agent that would need to be presentin a given vessel before a substantial difference in attenuation couldbe measured.

[0030] Measurement of the attenuation contrast caused by microspheres ofAlbunex (Molecular Biosystems, San Diego, Calif.) in vitro has beenaccomplished and it has been suggested that in vivo attenuation contrastmeasurement could be achieved. H. Bleeker, K. Shung, J. Burnhart, “Onthe Application of Ultrasonic Contrast Agents for Blood Flowometry andAssessment of Cardiac Perfusion,” J. Ultrasound Med. 9:461-471 (1990).Albunex is a suspension of 2-4 micron encapsulated air-filledmicrospheres that have been observed to have acceptable stability invivo and are sufficiently small in size that contrast enhancement canoccur in the left atrium or ventricle. Also, attenuation contrastresulting from iodipamide ethyl ester (IDE) particles accumulated inthe-liver has been observed. Under such circumstances, the contrastenhancement is believed to result from attenuation of the ultrasoundwave resulting from the presence of dense particles in a soft medium.The absorption of energy by the particles occurs by a mechanism referredto as “relative motion.” The change in attenuation caused by relativemotion can be shown to increase linearly with particle concentration andas the square of the density difference between the particles and thesurrounding medium. K. J. Parker, et al., “A Particulate Contrast Agentwith Potential for Ultrasound Imaging of Liver,” Ultrasound in Medicine& Biology, Vol. 13, No. 9, p. 555, 561 (1987). Therefore, wheresubstantial accumulation of solid particles occurs, attenuation contrastmay be a viable mechanism for observing image contrast enhancementalthough the effect is of much smaller magnitude than the backscatterphenomenon and would appear to be of little use in cardiovasculardiagnoses.

[0031] C. Speed of Sound Differential

[0032] An additional possible technique to enhance contrast in anultrasound image has been proposed, based on the fact that the speed ofsound varies depending on the media through which it travels. Therefore,if a large enough volume of an agent, through which the speed of soundis different than the surrounding tissue, can be infused into a targetarea, the difference in the speed of sound through the target area maybe measurable. Presently, this technique is only experimental.

[0033] Therefore, considering the three techniques described above forcontrast enhancement in an ultrasound image, the marked increase inbackscatter caused by free gas microbubbles is the most dramatic effectand contrast-enhancing agents that take advantage of this phenomenonwould be the most desirable if the obstacle of their limited stabilityin solution could be overcome.

The Materials Presently used as Contrast-Enhancing Agents

[0034] In light of what is known about the various techniques describedabove, attempts to develop a contrast-enhancing agent whose presencegenerates substantial contrast in an ultrasound image, and whosesurvival in vivo is sufficiently long to allow contrast-enhanced imagingof the cardiovascular system, has led to the investigation of a broadvariety of substances—gases, liquids, solids, and combinations ofthese—as potential contrast-enhancing agents.

[0035] A. Solid Particles

[0036] Typically, the solid substances that have been studied aspotential contrast-enhancing agents are extremely small particles thatare manufactured in uniform size. Large numbers of these particles canbe infused and circulate freely in the bloodstream or they may beinjected into a particular structure or region in the body.

[0037] IDE particles are solid particles that can be produced in largequantities with a relatively narrow size distribution of approximately0.5-2.0 microns. Sterile saline injections of these particles may beinjected and will tend to accumulate in the liver. Once a substantialaccumulation occurs, contrast enhancement may be exhibited by eitherattenuation contrast or backscatter mechanisms. Although suspensionscomprising these solid particles dispersed in a liquid may exhibitacceptable stability, the backscatter or attenuation effects arerelatively minor compared to free gas bubbles and a substantialaccumulation of the particles must occur before appreciable contrast isobserved in an ultrasound image. Thus, use of these suspensions has beenlimited to certain cell types in which the particles have the tendencyto coagulate because unless the suspension becomes highly concentratedin particular tissue, the contrast enhancement will be minor.

[0038] SHU-454 (Schering, A. G., West Berlin, Germany) is anexperimental contrast-enhancing agent in powder form that, when mixedwith a saccharide diluent, forms a suspension of crystals of variousrhomboid and polyhedral shapes ranging in size from 5 to 10 microns.Although the precise mechanism by which these crystals enhanceultrasound contrast is not completely understood, it is suspected thatthe crystals may trap microbubbles in their structure or that thecrystals themselves may backscatter ultrasound energy by an as-yetundetermined mechanism.

[0039] B. Liquids and Emulsions

[0040] In another attempt to achieve a satisfactory agent, emulsions areprepared by combining a chemical species compatible with body tissue anda species that provides high ultrasound contrast enhancement. EuropeanPatent Application 0231091 discloses emulsions of oil in watercontaining highly fluorinated organic compounds that have been studiedin connection with their possible use as a blood substitute and are alsocapable of providing enhanced contrast in an ultrasound image.

[0041] Emulsions containing perfluorooctyl bromide (PFOB) have also beenexamined. Perfluorooctyl bromide emulsions are liquid compounds known tohave the ability to transport oxygen. PFOB emulsions have exhibited alimited utility as ultrasound contrast agents because of a tendency toaccumulate in certain types of cells. Although the mechanism is notcompletely understood, PFOB emulsions may provide ultrasound contrastbecause of their high density and relatively large compressibilityconstant.

[0042] U.S. Pat. No. 4,900,540 describes the use of phospholipid-basedliposomes containing a gas or gas precursor as a contrast-enhancingagent. A liposome is a microscopic, spherical vesicle, containing abilayer of phospholipids and other amphipathic molecules and an inneraqueous cavity, all of which is compatible with the cells of the body.In most applications, liposomes are used as a means to encapsulatebiologically active materials. The above reference discloses the use ofa gas or gas precursors incorporated into the liposome core to provide alonger life span for the gas when infused into the body. Production ofstable liposomes is an expensive and time consuming process requiringspecialized raw materials and equipment.

[0043] C. Microbubbles

[0044] As noted above, a critical parameter that must be satisfied by amicrobubble used as a contrast- enhancing agent is size. Free gasmicrobubbles larger than approximately 8 microns may still be smallenough to avoid impeding blood flow or occluding vascular beds. However,microbubbles larger than 8 microns are removed from the bloodstream whenblood flows through the lungs. As noted above, medical researchers havereported in the medical-literature that microbubbles small enough topass through the lungs will dissolve so quickly that contrastenhancement of left heart images is not possible with a free gasmicrobubble. Meltzer, R. S., Tickner, E. G., Popp, R. L., “Why Do theLungs Clear Ultrasonic Contrast?” Ultrasound in Medicine and Biology.vol. 6, p.263, 267 (1980).

[0045] However, cognizant of the advantages to be gained by use ofmicrobubbles as contrast-enhancing agents by virtue of their largescattering cross-section, considerable attention has been focused ondeveloping mixtures containing microbubbles that are rendered stable insolution. Enhancing the stability of gas microbubbles may beaccomplished by a number of techniques.

[0046] Each of the following techniques essentially involves suspendinga collection of microbubbles in a substrate in which a bubble ofordinary gas is more stable than in the bloodstream.

[0047] In one approach, microbubbles are created in viscous liquids thatare injected or infused into the body while the ultrasound diagnosis isin progress. The theory behind the use of viscous fluids involves anattempt to reduce the rate at which the gas dissolves into the liquidand, in so doing, provide a more stable chemical environment for thebubbles so that their lifetime is extended.

[0048] Several variations on this general approach have been described.EPO Application No. 0324938 describes a viscous solution of abiocompatible material, such as a human protein, in which microbubblesare contained. By submitting a viscous protein solution to sonication,microbubbles are formed in the solution. Partial denaturation of theprotein by chemical treatment or heat provides additional stability tomicrobubbles in the solution by decreasing the surface tension betweenbubble and solution.

[0049] Therefore, the above approaches may be classified as an attemptto enhance the stability of microbubbles by use of a stabilizing mediumin which the microbubbles are contained. However, none of theseapproaches have addressed the primary physical and chemical propertiesof gases which have seriously limited the use of free gas microbubblesin ultrasound diagnosis, particularly with respect to the cardiovascularsystem. None of these approaches suggest that selection of the gases, byprecise criteria, would yield the ability to produce stable microbubblesat a size that would allow transpulmonary contrast-enhanced ultrasoundimaging.

[0050] The behavior of microbubbles in solution can be describedmathematically based on certain parameters and characteristics of thegas of which the bubble is formed and the solution in which the bubbleis present. Depending on the degree to which a solution is saturatedwith the gas of which the microbubbles are formed, the survival time ofthe microbubbles can be calculated. P. S. Epstein, M. S. Plesset, “Onthe Stability of Gas Bubbles in Liquid-Gas Solutions,” The Journal ofChemical Physics, Vol. 18, n. 11, 1505 (1950). Based on calculations, itis apparent that as the size of the bubble decreases, the surfacetension between bubble and surrounding solution increases. As thesurface tension increases, the rate at which the bubble dissolves intothe solution increases rapidly and, therefore, the size of the bubbledecreases more and more rapidly. Thus, the rate at which the bubbleshrinks increases as the size of the bubble decreases. The ultimateeffect of this is that a population of small free gas microbubblescomposed of ordinary air dissolves so rapidly that thecontrast-enhancing effect is extremely short lived. Using knownmathematical formula, one can calculate that a microbubble of air thatis 8 microns in diameter, which is small enough to pass through thelungs, will dissolve in between 190 and 550 milliseconds depending onthe degree of saturation of the surrounding solution. Based on thesecalculations, medical investigators studying the way in which the lungsremove ultrasound contrast agent have calculated the dissolution timesof oxygen and nitrogen gas microbubbles in human and canine blood andhave concluded that free gas microbubble contrast agents will not allowcontrast-enhanced imaging of the left ventricle because of the extremelybrief life of the microbubbles.

[0051] The physical properties of the systems that feature gas bubblesor gases dissolved in liquid solutions have been investigated in detailincluding the diffusion of air bubbles formed in the cavitating flow ofa liquid and the scatter of light and sound in water by gas bubbles.

[0052] The stability of gas bubbles in liquid-gas solution has beeninvestigated both theoretically, Epstein P. S. and Plesset M. S., On theStability of Gas Bubbles in Liquid-Gas Solutions, J. Chem. Phys.18:1505-1509 (1950) and experimentally, Yang W J, Dynamics of GasBubbles in Whole Blood and Plasma, J. Biomech 4:119-125 (1971); Yang WJ, Echigo R., Wotton D R, and Hwang J B, Experimental Studies of theDissolution of Gas Bubbles in Whole Blood and Plasma-I. StationaryBubbles. J. Biomech 3:275-281 (1971); Yang W J, Echigo R., Wotton D R,Hwang J B, Experimental Studies of the Dissolution of Gas Bubbles inWhole Blood and Plasma-II. Moving Bubbles or Liquids. J. Biomech4:283-288 (1971). The physical and chemical properties of the liquid andthe gas determine the kinetic and thermodynamic behavior of the system.The chemical properties of the system which influence the stability of abubble, and accordingly the life time, are the rate and extent ofreactions which either consume, transform, or generate gas molecules.

[0053] For example, a well known reaction that is observed between a gasand a liquid takes place when carbon dioxide gas is present in water. Asthe gas dissolves into the aqueous solution, carbonic acid is created byhydration of the carbon dioxide gas. Because carbon dioxide gas ishighly soluble in water, the gas diffuses rapidly into the solution andthe bubble size diminishes rapidly. The presence of the carbonic acid inthe solution alters the acid-base chemistry of the aqueous solution and,as the chemical properties of the solution are changed by dissolution ofthe gas, the stability of the carbon dioxide gas bubbles changes as thesolution becomes saturated. In this system, the rate of dissolution of agas bubble depends in part on the concentration of carbon dioxide gasthat is already dissolved in solution.

[0054] However, depending on the particular gas or liquid present in thesystem, the gas may be substantially insoluble in the liquid anddissolution of a gas bubble will be slower. In this situation, it hasbeen discovered that it is possible to calculate bubble stability in agas-liquid system by examining certain physical parameters of the gas.

BRIEF DESCRIPTION OF THE INVENTION

[0055] It has been discovered that it is possible to identify chemicalsystems where extremely small gas bubbles are not reactive in an aqueoussolution. Relying on the method disclosed herein one skilled in the artmay specially select particular gases based on their physical andchemical properties for use in ultrasound imaging. These gases can beused to produce the contrast-enhancing media that is also the subjectmatter of this invention. The microbubbles can be produced using certainexisting techniques that use ordinary air, and can be infused as in aconventional ultrasound diagnosis.

[0056] The method that is the subject matter of this invention requiresthat calculations be made, consistent with the equations providedherein, based on the intrinsic physical properties of a gas and aliquid. Particularly, the density of a gas, the solubility of a gas insolution, and the diffusivity of a gas in solution, which in turn isdependent on the molar volume of the gas and the viscosity of thesolution, are used in the equations disclosed below. Thus, by the methoddisclosed herein, the physical properties of a given gas-liquid systemcan be evaluated, the rate and extent of bubble collapse can beestimated, and gases that would constitute effective contrast-enhancingagents can be selected based on these calculations. Using existingtechniques, substantially improved contrast-enhancing media may then beproduced and used to improve the quality and usefulness of ultrasoundimaging.

DETAILED DESCRIPTION OF THE INVENTION

[0057] To understand the method of this invention, it is useful toderive the mathematical relationships that describe the parameters of agas-liquid system and the effect on bubble stability that occurs when avalue for one or more of these parameters is altered. It is assumedthat, at an initial time, T₀, a spherical gas bubble of gas X, with aradius of R₀, is placed in a solution in which the initial concentrationof gas X dissolved in the solution is equal to zero. Over some period oftime, the bubble of gas X will dissolve into the solvent at which timeits radius R will equal zero. Assume further that the solution is atconstant temperature and pressure and that the dissolved gasconcentration for a solution saturated with the particular gas isdesignated C_(s). Thus, at T_(o), the concentration of the gas in thesolution is zero, meaning that none of the gas has yet dissolved and allof the gas that is present is still contained within the bubble ofradius R₀.

[0058] As time progresses, due to the difference in the concentration-ofthe gas in the bubble and the gas in solution, the bubble will tend toshrink as gas in the bubble is dissolved into the liquid by the processof diffusion. The change in bubble radius from its original radius of R₀to, after the passage of a particular amount of time, a smaller radius Ris expressed by Equation (1),$\frac{R}{R_{0}} = \left\lbrack {1 - {\left( \frac{2{DC}_{s}}{\rho \quad R_{0}^{2}} \right)T}} \right\rbrack^{1/2}$

[0059] where R is the bubble radius at time T, D is the coefficient ofdiffusivity of the particular gas in the liquid, and ρ is the density ofthe particular gas of which the bubble is composed.

[0060] It follows that the time T required for a bubble to dissolvecompletely may be determined from Equation (1) by setting R/R₀=0, andsolving for T: $\begin{matrix}{T = \frac{R_{0}^{2}\rho}{2\quad D\quad C_{s}}} & {{Equation}\quad (2)}\end{matrix}$

[0061] This result qualitatively indicates that bubble stability, andhence life span, is enhanced by increasing the initial bubble size R₀ orby selecting a gas of higher density ρ, lower solubility C_(s) in theliquid phase, or a lower coefficient of diffusivity D.

[0062] The diffusivity D of a gas in a liquid is dependent on the molarvolume of the gas (Vm), and the viscosity of the solution (η) asexpressed by a known Equation;

D=13.26×10⁻⁵·η^(−1.14) ·V _(m) ^(−0.589)

[0063] By substituting the expression for D given in Equation (3) intoEquation (2) it is revealed that bubble stability is enhanced by usinggases of larger molar volume Vm, which tend to have a higher molecularweight, and liquids of higher viscosity.

[0064] By way of example, a comparison of the stability of airmicrobubbles and microbubbles composed of gases specially selected bythe method disclosed herein may be made. Taking the value of D for airin water at 22° C. as 2×10⁻⁵ cm²sec⁻¹ and the ratio C_(s)/ρ=0.02(Epstein and Plesset, Ibid.), one obtains the following data for thetime t for complete solution of air bubbles in water (unsaturated withair) : TABLE I INITIAL BUBBLE DIAMETER, microns TIME, milliseconds 12450 10 313 8 200 6 113 5 78 4 50 3 28 2 13 1 3

[0065] If the blood transit time from the pulmonary capillaries to theleft ventricle is two seconds or more (Hamilton, W. F. editor, Handbookof Physiology, Vol. 2, section 2, CIRCULATION. American PhysiologySociety, Wash., D.C., p. 709, (1963)), and recognizing that onlymicrobubbles of approximately 8 microns or less will be small enough topass through the lungs, it is clear that none of these bubbles have alife span in solution long enough to be useful contrast agents forultrasound contrast-enhanced imaging of the left ventricle.

[0066] The method of the present invention allows identification ofpotentially useful gases by comparing the properties of any particulargas, denoted gas X in the following description, to air. TakingEquations (2) and (3) above, a coefficient Q may be formulated for aparticular gas X that will describe the stability of microbubblescomposed of gas X in a given liquid. The value of the Q coefficientdetermined by this method for a particular gas X also can be used todetermine the utility of gas X as an ultrasound contrast-enhancing agentas compared to ordinary air.

[0067] From Equation (2) above, an equation that describes the time forcomplete dissolution of a bubble of gas X compared to the same sizebubble of ordinary air under identical conditions of solutiontemperature and solution viscosity may be written based on the physicalproperties of gas X and air: $\begin{matrix}{T_{x} = {{{T_{air}\left\lbrack \frac{\rho_{x}}{\rho_{air}} \right\rbrack}\quad\left\lbrack \frac{\left( C_{s} \right)_{air}}{\left( C_{s} \right)_{x}} \right\rbrack}\quad\left\lbrack \frac{D_{air}}{13.26 \times {10^{- 5} \cdot \eta^{- 1.14} \cdot \left( v_{m} \right)_{x}^{- {.589}}}} \right\rbrack}} & {{Equation}\quad (4)}\end{matrix}$

[0068] or, if D is known for gas X, $\begin{matrix}{T_{x} = {{{T_{air}\left\lbrack \frac{{\rho \quad}_{x}}{\rho_{air}} \right\rbrack}\quad\left\lbrack \frac{\left( C_{s} \right)_{air}}{\left( C_{s} \right)_{x}} \right\rbrack}\quad\left\lbrack \frac{D_{air}}{D_{x}} \right\rbrack}} & {{Equation}\quad (5)}\end{matrix}$

[0069] To formulate this equation so that the value Q may be obtained toenable comparison of gas X with air, the above equation may berewritten: $\begin{matrix}{T_{x} = {{{QT}_{air}\quad {where}\quad Q} = {{\left\lbrack \frac{\rho_{x}}{\rho_{air}} \right\rbrack \quad\left\lbrack \frac{\left( C_{s} \right)_{air}}{\left( C_{s} \right)_{x}} \right\rbrack}\quad\left\lbrack \frac{D_{air}}{D_{x}} \right\rbrack}}} & {{Equation}\quad (6)}\end{matrix}$

[0070] Assuming for comparison, a solution of water at 22 degrees C.,the density, diffusivity, and solubility of air in the solution areknown quantities which may be substituted into the above equationyielding: $\begin{matrix}{Q = {4.0 \times {10^{- 7}\left\lbrack \frac{\rho_{x}}{\left( C_{x} \right)_{x}D_{x}} \right\rbrack}}} & {{Equation}\quad (7)}\end{matrix}$

[0071] Substituting Equation (3) into the above for gases whosediffusivity D_(x) is not readily known, and assuming that the viscosityterm η below for water at 22 degrees C. is approximately equal to 1.0cP, $\begin{matrix}{Q = {3.0 \times {10^{- 3}\left\lbrack \frac{\rho_{x}}{\left( C_{s} \right)_{x}\left( V_{m} \right)_{x}^{- {.589}}} \right\rbrack}}} & {{Equation}\quad (8)}\end{matrix}$

[0072] Thus, knowing the density, solubility and molar volume of a gas,this method allows the calculation of the value of the Q coefficient.

[0073] If Q is less than one, microbubbles of gas X will be less stablein a given solvent than microbubbles of air. If Q is greater than one,microbubbles formed of gas X are more stable than microbubbles of airand will survive in solution longer than air bubbles. All otherproperties being the same for a given microbubble size, the time forcomplete dissolution of a microbubble of gas X is equal to the time forcomplete dissolution of a microbubble of ordinary air multiplied by theQ coefficient. For example, if the Q coefficient for gas X is 10,000, amicrobubble of gas X will survive 10,000 times as long in solutioncompared to a microbubble of air. A Q value can be determined for anygas in any solution assuming the quantities identified herein are knownor can be estimated.

[0074] Different methods for determining or estimating values for theindividual parameters of density, diffusivity, and solubility may beneeded depending on the chemical structure of the gas. Values for theseparameters may or may not be available from known scientific literaturesources such as the Gas Encyclopedia or the tabulations published by theAmerican Chemical Society. Values for the density of most gases arereadily available from sources such as the Handbook of Chemistry andPhysics, CRC Press, 72d Ed. (1991-92). Additionally, the solubility inwater and molar volume of some gases has been measured with accuracy. Inmany cases however, calculations for the numerical values for molarvolume and solubility may need to be calculated or estimated to providethe data used to determine the value of the Q coefficient for anindividual gas by the method described above. An example of thecalculation of Q values for a preferred selection of gases illustrateshow the method of this invention can be applied to individual gases.

[0075] Generally, many fluorine-containing gases exhibit extremely lowsolubility in water, and have relatively high molecular weights, highmolar volumes, and high densities. To determine the Q value for severalgases, the solubility, molar volume and density of the individual gasesare determined and the values are substituted into Equations (7) or (8)above.

[0076] Determination of Gas Solubility for Fluorocarbons

[0077] This method for estimating the gas solubility of fluorocarbonsuses extrapolation of the experimental data of Kabalnov A S, Makarov KN, and Scherbakova O V. “Solubility of Fluorocarbons in Water as a KeyParameter Determining Fluorocarbon Emulsion Stability,” J. Fluor. Chem.50, 271-284, (1990). The gas solubility of these fluorocarbons isdetermined relative to perfluoro-n-pentane which has a water solubilityof 4.0×10⁻⁶ moles per liter. For a homologous series of non-branchedfluorocarbons, the gas solubility may be estimated by increasing orreducing this value by a factor of about 8.0 for each increase orreduction in the number of additional —CF₂— groups present in themolecule.

[0078] Determination of Molar Volume

[0079] The molar volume (Vm) is estimated from the data of Bondi A.,“Van der Waals Volumes and Radii,” J. Phys. Chem. 68, 441-451 (1964).The molar volume of a gas can be estimated by identifying the number andtype of atoms that make up the molecule of gas in question. Bydetermining the number and type of atoms present in the molecule and howthe individual atoms are bound to each other, known values may beapplied for the molecular volume of the individual atoms. By consideringthe contribution of each individual atom and its frequency ofoccurrence, one may calculate the total molar volume for a particulargas molecule. This calculation is best demonstrated with an example.

[0080] It is known that a carbon molecule in an alkane carbon-carbonbond has a molar volume of 3.3 cubic centimeters per mole, a carbon atomin an alkene carbon-carbon bond has a molar volume of 10.0 cubiccentimeters per mole, and when multiple fluorine atoms are bound to analkane carbon, a fluorine atom has a molar volume of 6.0 cubiccentimeters per mole.

[0081] Examining octafluoropropane, this molecule contains three carbonatoms in alkane carbon-carbon bonds (3 atoms at 3.3 cubic centimetersper mole) and 6 fluorine atoms bound to alkane carbons (6 atoms at 6.0cubic centimeters per mole) hence, octafluoro-propane has a molardensity of 58 cubic centimeters per mole.

[0082] Once density, molar volume, and solubility are determined, the Qvalue is calculated using Equation 8 above.

[0083] The following Table lists the Q value for a number of gases basedon the calculations detailed above. TABLE II SOLUBILITY MOLAR DENSITYmicromoles/ VOLUME GAS kg/m3 liter cm3/mole Q Argon 1.78 1500 17.9 20n-Butane 2.05 6696 116 5 Carbon Dioxide 1.98 33000 19.7 1Decafluorobutane 11.21 32 73 13,154 Dodecafluoro- 12.86 4 183 207,437pentane Ethane 1.05 2900 67 13 Ethyl ether 2.55 977,058 103 0.1 Helium0.18 388 8 5 Hexafluorobuta-1, 9 (*) 2000 56 145 3-diene Hexafluoro-2- 9(*) 2000 58 148 butyne Hexafluoroethane 8.86 2100 43 116Hexafluoropropane 10.3 260 58 1299 Krypton 3.8 2067 35 44 Neon 0.90 43417 33 Nitrogen ## ## ## 1 Octafluoro-2-butene 10 (*) 220 65 1594Octafluoro- 9.97 220 61 1531 cyclobutane Octafluoropropane 10.3 260 581299 Pentane 2 1674 113 58 Propane 2.02 2902 90 30 Sulfur Hexafluoride5.48 220 47 722 Xenon 5.90 3448 18 28 # of 2 × 10⁻⁵ cm²sec-1 given abovewere used in equation 7 for this Q-value # determination.

[0084] Once the Q value has been determined the utility of an individualgas as an ultrasound contrast-enhancing agent can be analyzed bydetermining the life span of a collection of microbubbles composed ofthe gas in question at different sizes, as was done for air in Table Iabove. Taking the value of Q for decafluorobutane and examining the timenecessary for various sized bubbles to dissolve in water, one obtainsthe values in Table III below by multiplying each of the time values inTable I by the Q value for decafluorobutane: TABLE III INITIAL BUBBLEDIAMETER, microns TIME, minutes 12 99 10 69 8 44 6 25 5 17 4 11 3 6.1 22.9 1 0.7

[0085] Notice that the time scale in Table III is minutes rather thanmilliseconds as was the case for air. All bubbles of decafluorobutane,even as small as 1 micron, can be injected peripherally and will notdissolve into solution during the approximately 10 seconds needed toreach the left ventricle. Similar calculations can be performed for agas with any Q coefficient. Slightly larger bubbles will be able to passthrough the lungs and yet survive long enough to permit both examinationof myocardial perfusion and dynamic abdominal organ imaging. Moreover,as with many of the gases identified by this method, decafluorobutanefeatures low toxicity at small dosages and would, therefore, offersubstantial advantages as a contrast-enhancing agent in conventionalultrasound diagnosis.

[0086] Manual creation of a microbubble suspension may be accomplishedby several methods. U.S. Pat. No. 4,832,941, the disclosure of which isincorporated herein by reference, refers to a method for producing asuspension of microbubbles with a diameter less than seven micronscreated by spraying a liquid through a quantity of gas using a three-waytap. Although techniques could vary in practice, the three-way tap is apreferred method to manually suspend a quantity of high Q coefficientgas to produce the contrast-enhancing media described herein.

[0087] The general techniques for use of a three-way tap device are wellknown in connection with preparation of the common Freund's adjuvant forimmunizing research animals. Typically, a three-way tap is comprised ofa pair of syringes, both of which are connected to a chamber. Thechamber has outlet from which the suspension may be collected or infuseddirectly.

[0088] Techniques for use of the three-way tap may differ from thatdescribed in U.S. Pat. No. 4,832,941 because different gases are beingused in this procedure. For example, use of one of the high Qcoefficient gases disclosed herein may be more efficient if the systemis purged of ordinary air or flushed with another gas before themicrobubble suspension is produced.

[0089] In a preferred embodiment of the present invention, a 40-50%Sorbitol (D-glucitol) solution is mixed with approximately 1-10% byvolume of a high Q-coefficient gas with approximately 5% gas being anoptimal value. Sorbitol is a commercially available compound that whenmixed in an aqueous solution substantially increases the viscosity ofthe solution. Higher viscosity solutions, as seen in equation 3 above,extend the life of a microbubble in solution. A 40-50% Sorbitol solutionis preferred to maintain as a bolus upon injection; that is as intact aspossible without exceeding a tolerable injection pressure. To producethe suspension of microbubbles, a quantity of the chosen gas iscollected in a syringe. In the same syringe, a volume of the Sorbitolsolution may be contained. A quantity of Sorbitol solution is drawn intothe other syringe so that the sum of the two volumes yields the properpercentage of gas based on the volume percentage of microbubblesdesired. Using the two syringes, each featuring a very small aperture,the liquid is sprayed into the gas atmosphere approximately 25 times oras many times as is necessary to create a suspension of microbubbleswhose size distribution is acceptable for the purposes described herein.This technique may be varied slightly, of course, in any manner thatachieves the resulting suspension of microbubbles of the desired size ina desired concentration. Microbubble size may be checked either visuallyor electronically using a Coulter Counter (Coulter Electronics) by aknown method, Butler, B. D., “Production of Microbubble for Use as EchoContrast Agents,” J. Clin. Ultrasound, V.14 408 (1986).

EXAMPLES Example 1

[0090] An ultrasound contrast agent was prepared using decafluorobutaneas the microbubble-forming gas. A solution was prepared containing:sorbitol 20.0 g NaCl 0.9 g soy bean oil 6.0 mL Tween 20 0.5 mL waterq.s. 100.0 mL

[0091] A soapy, clear, yellow solution was afforded with stirring. A 10mL aliquot of this solution was taken up in a 10 mL glass syringe. Thesyringe was then attached to a three-way stopcock. A second 10 mLsyringe was attached to the stopcock and 1.0 cc of decafluorobutane(PCR, Inc., Gainesville, Fla.) was delivered to the empty syringe. Thestopcock valve was opened to the solution-containing syringe and theliquid and gas phases mixed rapidly 20-30 times. A resultingmilky-white, slightly viscous solution was obtained.

Example 2

[0092] The gas emulsion obtained in Example 1 was diluted with water(1:10 to 1:1000), placed in a hemocytometer, and examined under themicroscope using an oil immersion lens. The emulsion consisted ofpredominately 2-5 micron bubbles. The density was 50-100 millionmicrobubbles per mL of original undiluted formulation.

Example 3

[0093] The formulation of Example 1 was prepared and echocardiographyperformed in a canine model. A 17.5 kg mongrel dog was anesthetized withisoflurane and monitors established to measure ECG, blood pressure,heart rate, and arterial blood gases according to the methods describedby Keller, M W, Feinstein, S B, Watson, D D: Successful left ventricularopacification following peripheral venous injection of sonicatedcontrast agent: An experimental evaluation. Am Heart J 114:570d (1987).

[0094] The results of the safety evaluation are as follows:

Maximum Percentage Change in Measured Parameter Within 5 Min PostInjection

[0095] AORTIC PRESSURE SYS- DIAS- BLOOD TOLIC TOLIC GASES HEART DOSE mmHg MEAN PaO2 PaCO2 pH RATE 0.5 mL +6, −14 +9, 0   +8, −6 329 58.1 7.26+10 −19 1.0 mL +9, −2  +5, −1 +4, −4 +1, −4 2.0 mL +5, −3  +5, −1 +5, −1  0, −1 3.0 mL +6, −2  +7, 0   +4, −3   0, −3 4.0 mL +5, −1  +3, −3 +5,−3   0, −3 5.0 mL   0, −10 +1, −3   0, −4 +1, −1 7.0 mL   0, −13   0, −8  0, −9 313 28.6 7.36   0, −1

[0096] All changes were transient and returned to baseline valuestypically within 3-6 minutes. The above safety data demonstrate minimalchanges in the measured hemodynamic parameter. All doses provided bothright and left ventricular chamber opacification. The intensityincreased with the increasing dose.

Example 4

[0097] The above specific determinations of the suitability of aparticular gas for use as an ultrasound agent can be approximated if themolecular weight of a particular gas is known, can be calculated, or canbe measured. This approximation is based on the determination that thereis a linear relationship between the logarithm of the Q-value and themolecular weight for a gas, as shown in the Figure below.

[0098] Based on this Figure, the following guidelines can be used toestimate a Q-value: Molecular Weight Estimated Q-Value  <35 <5 35-70 5-20  71-100 21-80 101-170  81-1000 171-220   1001-10,000 221-270 10,001-100,000 >270 >100,000

[0099] The following Table contains a series of gases with the relevantdata on molecular weight and estimated Q-value. The higher the Q-valuethe more promising is the particular gas. Especially promising are gaseswith Q-values greater than five. Additional issues, including, but notlimited to, cost and toxicity, should be considered in addition tolongevity of the derived microbubbles (as estimated by the Q-value) indetermining the suitability of any particular gas as an ultrasoundcontrast agent. TABLE IV Molecular Estimated Q Chemical Name WeightValue Acetone, hexafluoro 166.02  81-1000 Acetylene, isopropyl 68  5-20Air 28.4 <5 Allene 40.06  5-20 Allene, tetrafluoro 112.03  81-1000 Argon39.98  5-20 Borne, dimethyl, methoxy 71.19 21-80 Borne, trimethyl 55.91 5-20 Boron fluoride dihydrate 103.84  81-1000 1,2-Butadiene 54.09  5-201,3-Butadiene 54.09  5-20 1,3-Butadiene, 1,2,3-trichloro 157.43  81-10001,3-Butadiene, 2-fluoro 72.08 21-80 1,3-Butadiene, 2-methyl 68.12  5-201,3-Butadiene, hexafluoro 162.03  81-1000 Butadiyne 50.06  5-20 n-Butane58.12  5-20 Butane, 1-fluoro 76.11 21-80 Butane, 2-methyl 72.15 21-80Butane, decafluoro 238.03  10,001-100,000 1-Butene 56.11  5-20 2-Butene{cis} 56.11  5-20 2-Butene {trans} 56.11  5-20 1-Butene, 2-methyl 70.13 5-20 1-Butene, 3-methyl 70.13  5-20 2-Butene, 3-methyl 68  5-201-Butene, perfluoro 200.03   1001-10,000 2-Butene, perfluoro 200.03 1001-10,000 3-Butene-2-one, 4-phenyl {trans} 146.19  81-10001-Butene-3-yne, 2-methyl 66.1  5-20 Butyl nitrite 103.12  81-1001-Butyne 54.09  5-20 2-Butyne 54.09  5-20 Butyne,2-chloro-1,1,1,4,4,4-hexafluoro 199   1001-10,000 1-Butyne, 3-methyl68.12  5-20 2-Butyne, perfluoro 162.03  81-1000 Butyraldehyde, 2-bromo151  81-1000 Carbon dioxide 44.01  5-20 Carbonyl sulfide 60.08  5-20Crotononitrile 67.09  5-20 Cyclobutane 56.11  5-20 Cyclobutane, methyl70.13  5-20 Cyclobutane, octafluoro 200.03   1001-100,000 Cyclobutene,perfluoro 162.03  81-1000 Cyclopentene, 3-chloro 102.56  81-1000Cyclopropane 42.08  5-20 Cyclopropane, 1,2-dimethyl {trans, dl} 70.13 5-20 Cyclopropane, 1,1-dimethyl 70.13  5-20 Cyclopropane, 1,2-dimethyl{cis} 70.13  5-20 Cyclopropane, 1,2-dimethyl {trans, 1} 70.13  5-20Cyclopropane, ethyl 70.13  5-20 Cyclopropane, methyl 56.11  5-20Deuterium 4.02 <5 Diacetylene 50.08  5-20 Diaziridine, 3-ethyl-3-methyl86.14 21-80 Diazoethane, 1,1,1-trifluoro 110.04  81-1000 Dimethyl amine45.08  5-20 Dimethyl amine, hexafluoro 153.03  81-1000 Dimethyldisulfide, hexafluoro 202.13   1001-10,000 Dimethylethylamine 73.1421-80 bis-(Dimethyl phosphino) amine 137.1  81-10002,3-Dimethyl-2-norbornano 140.23  81-1000 Dimethylamine, perfluoro171.02   1001-10,000 Dimethyloxonium chloride 82.53 21-801,3-Dioxolane-2-one, 4-methyl 102.09  81-1000 Ethane 30.07 <5 Ethane,1,1,1,2-tetrafluoro 102.03  81-1000 Ethane, 1,1,1-trifluoro 84.04 21-80Ethane, 1,1,2,2-tetrafluoro 102.03  81-1000 Ethane,1,1,2-trichloro-1,2,2-trifluoro 187.38   1001-10,000 Ethane,1,1-dichloro 98 21-80 Ethane, 1,1-dichloro-1,2,2,2-tetrafluoro 170.92  1001-10,000 Ethane, 1,1-dichloro-1-fluoro 116.95  81-1000 Ethane,1,1-difluoro 66.05  5-20 Ethane, 1,2-dichloro-1,1,2,2-tetrafluoro 170.92  1001-10,000 Ethane, 1,2-difluoro 66.05  5-20 Ethane,1-chloro-1,1,2,2,2-pentafluoro 154.47  81-1000 Ethane,1-chloro-1,1,2,2-tetrafluoro 136.48  81-1000 Ethane,2-chloro,1,1-difluoro 100 21-80 Ethane, 2-chloro-1,1,1-trifluoro 118.49 81-1000 Ethane, Chloro 64.51  5-20 Ethane, chloro pentafluoro 154.47 81-1000 Ethane, dichlorotrifluoro 152  81-1000 Ethane, fluoro 48.06 5-20 Ethane, hexafluoro 138.01  81-1000 Ethane, nitro-pentafluoro165.02  81-1000 Ethane, nitroso-pentafluoro 149.02  81-1000 Ethane,perfluoro 138.01  81-1000 Ethyl amine, perfluoro 171.02   1001-100,000Ethyl ether 74.12 21-80 Ethyl methyl ether 60.1  5-20 Ethyl vinyl ether72.11 21-80 Ethylene 28.05 <5 Ethylene, 1,1-dichloro 96.94 21-80Ethylene, 1,1-dichloro-2-fluoro 114.93  81-1000 Ethylene,1,2-dichloro-1,2-difluoro 132.92  81-1000 Ethylene, 1,2-difluoro 64 5-20 Ethylene, 1-chloro-1,2,2-trifluoro 116.47  81-1000 Ethylene,chloro trifluoro 116.47  81-1000 Ethylene, dichloro difluoro 132.92 81-1000 Ethylene, tetrafluoro 100.02 21-80 Fulvene 78.11 21-80 Helium 4<5 1,5-Heptadiyne 92.14 21-80 Hydrogen (H2) 2.02 <5 Isobutane 58.12 5-20 Isobutane, 1,2-epoxy-3-chloro 106.55  81-1000 Isobutylene 56.11 5-20 Isoprene 68.12  5-20 Krypton 83.8 21-80 Methane 16.04 <5 Methanesulfonyl chloride, trifluoro 168.52  81-1000 Methane sulfonyl fluoride,trifluoro 152.06  81-1000 Methane, (pentafluorothio)trifluoro 196.06  1001-10,000 Methane, bromo difluoro nitroso 159.92  81-1000 Methane,bromo fluoro 112.93  81-1000 Methane, bromo-chloro-fluoro 147.37 81-1000 Methane, bromo-trifluoro 148.91  81-1000 Methane, chlorodifluoro nitro 131.47  81-1000 Methane, chloro dinitro 140.48  81-1000Methane, chloro fluoro 68.48  5-20 Methane, chloro trifluoro 104.46 81-1000 Methane, chloro-difluoro 86.47 21-80 Methane, dibromo difluoro209.82   1001-10,000 Methane, dichloro difluoro 120.91  81-1000 Methane,dichloro-fluoro 102.92  81-1000 Methane, difluoro 52.02  5-20 Methane,difluoro-iodo 177.92   1001-10,000 Methane, disilano 76.25 21-80Methane, fluoro 34.03 <5 Methane, iodo- 141.94  81-1000 Methane,iodo-trifluoro 195.91   1001-10,000 Methane, nitro-trifluoro 115.01 81-1000 Methane, nitroso-trifluoro 99.01 21-80 Methane, tetrafluoro 8821-80 Methane, trichlorofluoro 137.37  81-1000 Methane, trifluoro 70.01 5-20 Methanesulfenylchloride, trifluoro 136.52  81-1000 2-Methyl butane72.15 21-80 Methyl ether 46.07  5-20 Methyl isopropyl ether 74.12 21-80Methyl nitrite 61.04  5-20 Methyl sulfide 62.13  5-20 Methyl vinyl ether58.08  5-20 Neon 20.18 <5 Neopentane 72.15 21-80 Nitrogen (N2) 28.01 <5Nitrous oxide 44.01  5-20 1,2,3-Nonadecane tricarboxylic acid, 2-500.72 >100,000 . . . hydroxytrimethylester 1-Nonene-3-yne 122.21 81-1000 Oxygen (02) 32 <5 1,4-Pentadiene 68.12  5-20 n-Pentane 72.1521-80 Pentane, perfluoro 288.04 >100,000 2-Pentanone, 4-amino-4-methyl115.18  81-1000 1-Pentene 70.13  5-20 2-Pentene {cis} 70.13  5-202-Peatene {trans} 70.13  5-20 1-Pentene, 3-bromo 149.03  81-10001-Pentene, perfluoro 250.04  10,001-100,000 Phthalic acid, tetrachloro303.91 >100,000 Piperidine, 2,3,6-trimethyl 127.23  81-1000 Propane 44.1 5-20 Propane, 1,1,1,2,2,3-hexafluoro 152.04  81-1000 Propane, 1,2-epoxy58.08  5-20 Propane, 2,2-difluoro 80.08 21-80 Propane, 2-amino 59.11 5-20 Propane, 2-chloro 78.54 21-80 Propane, heptafluoro-1 -nitro 215.03  1001-10,000 Propane, heptafluoro-1 -nitroso 199.03   1001-10,000Propane, perfluoro 188.02   1001-10,000 Propene 42.08  5-20 Propyl,1,1,1,2,3,3-hexafluoro-2, 221  10,001-100,000 3-dichloro Propylene,1-chloro 76.53 21-80 Propylene, 1-chloro-{trans} 76.53  5-20 Propylene,2-chloro 76.53  5-20 Propylene, 3-fluoro 60.07  5-20 Propylene,perfluoro 150.02  81-1000 Propyne 40.06  5-20 Propyne, 3,3,3-trifluoro94.04 21-80 Styrene, 3-fluoro 122.14  81-1000 Sulfur hexafluoride 146.05 81-1000 Sulfur (di),decafluoro(S2F10) 298 >100,000 Toluene, 2,4-diamino122.17  81-1000 Trifluoroacetonitrile 95.02 21-80 Trifluoromethylperoxide 170.01  81-1000 Trifluoromethyl sulfide 170.07  81-1000Tungsten hexafluoride 298 >100,000 Vinyl acetylene 52.08  5-20 Vinylether 70  5-20 Xenon 131.29  81-1000

Example 5

[0100] The relationship between a calculated Q-value for a given gas andthe persistence of microbubbles of that gas was studied to determinewhat Q-value would be a lower limit for utility as an ultrasoundcontrast agent. For these experiments, a 190×100 mm Pyrex™ (No. 3140)evaporation dish was filled with approximately 2000 mL of water at 37°C. Five mL of a 20% sorbitol solution was taken up in a 10 mL syringeconnected to a three way stopcock. A 10 mL syringe, containing 2 cubiccentimeters of the subject gas (or low boiling liquid, as pertinent) wasattached to the syringe containing the sorbitol solution. The sorbitoland gas or liquid are rapidly mixed 25-times to create a suspension ofmicrobubbles or dispersed liquid and then rapidly added to the water.The microbubbles of this method are generally about 100 microns in size,and if composed of air would have a calculated persistence of 31 sec(0.5 min). Ultrasound scans before, during and after the addition weremade with a Hewlett-Packard Model Sonos 500 ultrasound scanner operatingat 5 MHz. The time during which the microbubbles could be observed wasrecorded. The results are contained in Table V below. The experimentalQ-values were obtained by dividing the measured persistence of a givengas by the measured persistence for air. TABLE V RELATIONSHIP BETWEENQ-VALUE FOR A GAS AND THE PERSISTENCE OF MICROBUBBLES PERSISTENCEQ-VALUE (Experimental GAS (Calculated) Q-Value) Diethyl ether 0.1 0.1min (0.2) Air 1 0.6 min (1.0) Butane 5 1.5 min (2.6) Helium 5 2.0 min(3.5) Propane 30 3.2 min (6.0) Pentane 58 20.6 min (36)Dodecafluoropentane 207,437 >5760 min (>10,105)

[0101] These experiments indicate an excellent agreement between thecalculated Q-value and the experimentally determined values. Based onthese data, gases with Q-values calculated to be greater than fiveshould be potentially useful as contrast agents for ultrasound imaging.

Example 6

[0102] The relationship of the state of matter of a given chemicalentity with a high Q-coefficient and its utility as an ultrasoundcontrast agent was tested by comparing the efficiency ofperfluoropentane and perfluorohexane to act as an ultrasound contrastagent. Perfluoropentane (dodecafluoropentane) has a calculatedQ-coefficient of 207,437 and a boiling point under standard pressureconditions of 29.5 degrees Centigrade. Perfluorohexane (PCR, INc.,Gainsville, Fla.) has a calculated Q-coefficient of 1,659,496 and aboiling point under standard pressure conditions of 59-60 degrees C.Therefore, at 37 degrees C., the body temperature of man,perfluoropentane is a gas while perfluorohexane is a liquid.

[0103] Aqueous dispersions of perfluoropentane and perfluorohexane (2%w/v) were formed at 4 degrees C. by vigorous homogenization. A plasticbeaker, containing approximately 1000 mL of water at 37 degrees C., wasprepared to simulate human blood and was ultrasonically-scanned, asindicated in Example 5 above, before and after the addition of samplesof each of the above dispersions.

[0104] Less than 1.0 mL of the perfluoropentane dispersion, when mixedwith the simulated blood, produced an extremely bright ultrasound signalwhich persisted for at least 30 minutes. A 1:10,000 dilution was stilldetectable.

[0105] In contrast, a 1.0 mL sample of the perfluorohexane dispersionwas undetectable by ultrasound scanning under the same conditions, aswas even a 10 mL sample (1:100 dilution).

[0106] The conclusion to be drawn is that both a high Q-coefficient anda gaseous state at the body temperature of the organism being scanned isnecessary for a substance to be effective as an ultrasound contrastagent according to the method that is the subject matter of thisinvention.

[0107] Although the invention has been described in some respects withreference to specified preferred embodiments thereof, variations andmodifications will become apparent to those skilled in the art. It is,therefore, the intention that the following claims not be given arestrictive interpretation but should be viewed to encompass variationsand modifications that are derived from the inventive subject matterdisclosed. In this regard the invention allows those skilled in the artto determine the suitability of various chemicals as ultrasound contrastagents where density, solubility and molar volume data are available.

I claim:
 1. Contrast media for ultrasound image-enhancement comprisingmicrobubbles of a biocompatible gas having a Q coefficient greater than5 where Q=4.0×10⁻⁷ ×ρ/C _(s) D and ρ is the density of the gas (Kgm⁻³),C_(s) is the water solubility of the gas (M) and D is the diffusivity ofthe gas in solution (cm³sec⁻¹)
 2. Contrast media of claim 1 comprising asuspension of gas bubbles smaller than 8 microns in a biocompatibleaqueous liquid vehicle.
 3. Contrast media of claim 1 wherein the gas issulfur hexafluoride.
 4. Contrast media of claim 1 wherein the gas ishexafluoropropylene.
 5. Contrast media of claim 1 wherein the gas isoctafluoropropane.
 6. Contrast media of claim 1 wherein the gas ishexafluoroethane.
 7. Contrast media of claim 1 wherein the gas isoctafluoro-2-butene.
 8. Contrast media of claim 1 wherein the gas ishexafluoro-2-butyne.
 9. Contrast media of claim 1 wherein the gas ishexafluorobuta-1,3-diene.
 10. Contrast media of claim 1 wherein the gasis octafluorocyclobutane.
 11. Contrast media of claim 1 wherein the gasis decafluorobutane.
 12. Contrast media of claim 1 where the gas isdodecafluoropentane.
 13. A method for selecting a gas for use as anultrasound image-enhancement agent comprising the steps of determiningthe solubility, C_(s), of the gas in a solution; determining thedensity, ρ, of the gas; determining the diffusivity, D, of the gas inthe solution; calculating a Q coefficient where Q=4.0×10⁻⁷ ×ρ/C _(s) Dand selecting a gas having a Q coefficient of greater than
 5. 14. Themethod of claim 1 wherein the diffusivity, D, is determined from themolar volume, Vm, of a gas by the formula D=13.26×10⁻⁵·η^(−1.14) ·V _(m)^(−0.589) where η is the solution viscosity (cP).